We propose to demonstrate the feasibility of a novel highly multiplexed targeted sequencing-based Liquid Biopsy TempO-Seq Assay to measure cell free circulating DNA (cfNA) from plasma samples, the cfNA TempO-Seq assay. We will exploit the single base specificity of TempO-Seq to measure SNVs and other mutations and measure microsatellite short tandem repeats and methylation with modifications to the standard TempO-Seq protocol. TempO-Seq has single cell sensitivity, which we will exploit to achieve the sensitivity to measure low abundant cfNA. We will use archived frozen plasma and matched FFPE obtained from normal subjects and colorecctal cancer patients available from the University of Arizona Cancer Center Biospecimens Repository, spiking synthetic targets and DNA purified from cancer cell lines into normal samples to establish sensitivity and reprooducibiolity and validate the method, then test archived plasma and matched FFPE samples from stage I, II, III, and IV colorectal cancer patients.